PREPARATION AND CHARACTERIZATION OF STAVUDINE ENTRAPPED LIPOSPHERES
Ranjita Shegokar* and Kamalinder Singh
C.U. Shah College of Pharmacy, S.N.D.T.
Women’s University, Santacruz (W), Mumbai- 400049 India.
Submitted: 12-07-2012 Revised: 29-09-2012 Accepted: 06-10-2012
*Corresponding author Ranjita Shegokar
Email :
ABSTRACT
Lipospheres of stavudine were prepared by melt dispersion technique using trimyristin, tripalmitin and triastearin, stearic acid, Compritol® 888 ATO and Precirol® ATO 5 as lipid matrix in the various drug-lipid ratios. Drug entrapped free flowing solid lipospheres of triglycerides and glyceryl behenate were characterized for surface morphology, particle size distribution, encapsulation efficiency, and in vitro release behavior. The effect of drug lipid ratio, the surfactant used, concentration of stabilizer, and stirring speed were identified as the key variables affecting the formation of discrete spherical lipospheres and sustained drug release rate. The lipospheres production conditions were optimized by using 2% w/w sodium cholate and 1% Plural oleic as a stabilizer.
The concentration of lipid used had pronounced effect on particle size of the lipospheres. The incorporation efficiency was found to be in range of 30 to 50%. Increase in concentration of surfactant and stirring speed produced fine spherical, smooth, and round lipospheres. All the prepared lipospheres exhibited slow release profiles dictating the Higuchi mode of release.
Key words: Lipospheres, stavudine, lipid matrix, hydrophilic surfactant, drug release
INTRODUCTION
Delivery systems are designed to protect an incorporated drug from the gastrointestinal (GI) environment during delivery and to provide a controlled release (Pandit et al., 2009).
The goal may be either to deliver a drug locally to specific sites in the body or to prepare a drug carrier system that acts as a reservoir at the site of injection over a certain period of time (Khopade et al., 1997; Nasr et al., 2008). During the last decade, numerous attempts were made to eradicate HIV virus, it was found that eradication of HIV is highly unlikely, and more effective antiretroviral therapy is required on a long-term basis to maintain viral suppression and reduce disease progression (Obaru and Mitsuva, 1998). Currently available anti-HIV drugs in marketed are in the form of solid dosage forms, (tablets and capsules) for oral use; or liquid dosage forms (e.g. solutions, suspensions) for oral use. However, conventional routes are inherently limited in that they cannot maintain a constant plasma level with the target therapeutic range for a prolonged duration. Oral drug delivery has its biggest disadvantage of first pass effect,
irrational of absorption and degradation in the gastrointestinal tract due to enzymes and extreme pH conditions, less duration of action with limited absorption of the drug depending on the mean residence time of the drug in GI tract (Alonso, 2004). Multi-drug therapy i.e.
HAART that attacks at several stages of HIV life cycle such as nucleotide reverse transcription, protease inhibition whereas therapeutic vaccine which can boost the immune response against the virus has failed because of mutating nature of virus.
Lipospheres are lipid micro particles in size range of 0.2 to 100µm, composed of solid hydrophobic fat matrix in which the bioactive compound are dissolved or dispersed.
Lipospheres offers advantage of good physical stability, low cost of ingredients and ease of preparation and scale up (Takenaga, 1996).
They have uniform particle structure, high encapsulation efficiency for hydrophobic drugs and slow release of entrapped drugs. In- vivo distribution of lipospheres has demonstrated a high affinity to vascular wells including capillaries, inflamed tissues and granulocytes (Khopade et al., 1997).
Lipospheres have successfully been used to incorporate and deliver a variety of substances, including anti-inflammatory compounds and local anesthetics antibiotics, insect repellants, vaccines and adjuvants (Pandit et al., 2009; Nasr et al., 2008; Attama et al., 2009; Bekerman et al., 2004; Cortesi et al., 2002
;
Elgart et al., 2012;
El-Gibaly and Abdel- Ghaffar,
2005;Hersh et al.,1992; Shivakumar et al., 2007; Sznitowska et al., 2000;
Toongsuwan et al., 2004; Tursilli et al., 2006; Unger et al., 1998; Vyas et al., 1997), only few peptide and protein drugs have been incorporated into lipospheres (Cortesi et al., 2002; Amselem et al., 1996).There is need to develop a suitable drug delivery system to combat complex dosing regimens, costs, side effects, biodistribution limitations, and variable drug pharmacokinetic patterns of antiretroviral. In this research, an attempt was made to formulate biocompatible stavudine lipospheres which will give prolonged drug release and might reduce side effects. Stavudine is a synthetic thymidine nucleoside analogue. Its triphosphate active metabolite inhibits HIV reverse transcriptase by competing with natural substrate thymidine triphosphate by causing DNA chain termination.
METHODOLOGY
Stavudine was obtained as gift sample from Alkem Laboratories, Mumbai, India Dynasan 114,116, 118 were generous gift from Sasol Germany. Compritol® ATO 888 Precirol® ATO 5 and Plurol® Oleique CC 497 were obtained as gift sample from Gattefosse, France through Colorcon Asia Pvt. Ltd., Goa, India. Surfactants such as Lutrol F68 and Tween80 were obtained from BASF and Uniquema, Mumbai, India, respectively. Stearic acid and Sodium cholate was purchased from S.D. Fine Chemicals, Mumbai, India.
Production of lipospheres
Drug encapsulated lipospheres were prepared by melt dispersion technique. Lipid solubility of stavudine was determined in selected lipids in presence and absence of surfactants. The lipid mixture containing
hydrophobic surfactant was melted at 700C and then emulsified into an external aqueous phase containing a hydrophilic surfactant. The emulsion was mechanically stirred for 30min at 700C under constant stirring at 6000rpm. At end, the milky emulsion was rapidly cooled at about 100C in ice bath for 15 minutes. The lipospheres were then washed with water and isolated by membrane filtration. The lipospheres obtained were then air dried overnight before characterization. Lipospheres were prepared using 2, 4 or 8%w/w of Dynasan 114,116, 118, Compritol® ATO 888, Precirol® ATO 5 and stearic acid. The optimum combination of four independent variables, such as concentration of lipid, concentration of surfactant, type of co- surfactant and stirring speed were varied at three levels to achieve maximum drug entrapment and optimum particle size (Table I).
Characterization of lipospheres
Lipospheres were evaluated for appearance, particle size, % drug entrapment and in-vitro drug release.
Particle size distribution
The particle size of all batches of stavudine loaded lipospheres was carried out using Hund Wetzlar image analyzer. The lipospheres were first dispersed in water and sonicated for 1min to obtain aggregate free suspension. A drop of this homogeneous suspension was placed on glass slide and observed under previously calibrated microscope. The photomicrographs were taken and particle size was measured for at least hundred particles and the average was calculated.
Percent drug entrapment
The drug-loading or entrapment of stavudine in lipid core was determined partitioning drug and lipid phase. Lipospheres were dissolved in separating flask containing 100mL of chloroform with vigorous shaking for 10min., followed by addition of water (100mL). Drug entrapped in lipospheres extracted in aqueous phase was determined by UV spectrophotometry at wavelength 265nm.
The percent drug entrapped was calculated by subtracting the obtained amount from total drug added.
In vitro drug release
In vitro drug release studies on selected batches were carried out using USP dissolution apparatus II at 50rpm. Phosphate buffer (pH 7.2) was medium used as dissolution medium maintained at 37±0.20C.
Aliquots were withdrawn at time intervals 30 min, 1, 2, 3, 4, 5, 6, 7, 8, and 24 h and filtered through whatman filter paper. A run for blank lipospheres was also performed.
The drug content in aliquots was determined using spectrophotometrically at 265nm.
Data analysis
The extent of drug release was assessed from the total amount of drug present in the
dissolution medium at the end of the 8 h drug release experiment. The type of drug release kinetics applicable for the Stavudine lipospheres was determined by evaluation of zero-order kinetic model (Q vs. t), where Q is the amount of drug released at time‘t’. The results expressed as mean ± SD were generated from replicate determinations for each suppository preparation.
Results and discussion
The influence of production parameters on the characteristics of lipospheres was studied. The melt dispersion technique produced white, free flowing, discrete spherical stavudine lipospheres. Out of six lipids investigated Dynasan 114, 116, 118 and Compritol® ATO 888 resulted in producing free flowing lipospheres as com- pared to stearic acid and Precirol® ATO 5.
Table I Experimental design applied for production of lipospheres lists the independent variables and their levels.
Levels Independent variables
1 2 3
A) Conc. of lipid (% w/w) 2 4 8
B) Type Co-surfactant (1% w/w) F68 Tween 80 sodium cholate
C) Conc. surfactant (% w/w) 1 2 5
D) Stirring speed 2000 4000 6000
Figure 1. Photomicrographs of lipospheres prepared using different lipids a) Dynasan 114 b) Dynasan 116 c) Dynasan 118 d) Compritol® ATO 888 e) Precirol® ATO 5 f) Stearic Acid.
Lipospheres obtained with stearic acid were irregular in shape while those prepared with Precirol® ATO 5 were sticky and agglomerated.(Fig 1) whereas lipospheres prepared with dynasan116 were more rigid and free flowing than Compritol® ATO 888.
Dynasan 114 and 118 produced slightly sticky but free flowing lipospheres.
The particle sizes of the lipospheres prepared with three dynasan’s were in the range of 15 to 40µm while for Precirol® ATO 5 and Compritol® 888 ATO were much
smaller (Fig 2). Triglycerides were able to entrap 50-55% of drug, while Compritol®
888 ATO and Precirol® ATO 5 could able to entrapped only 40% of drug. This clearly states that triglycerides have more drug entrapment than Compritol® 888 ATO, Precirol® ATO 5 and stearic acid.
The influence of preparation parameters, such as (a) type and amount of lipids, (b) presence and concentration of surfactants, (c) stirring speed was studied. The four selected independent variable s had marked effect on Figure 2 Effect of the type of lipid D4 (Dynasan 114), D6
(Dynasan 116), D8 (Dynasan 118), PRE (Precirol® ATO 5) and Com (Compritol® 888 ATO) on particle size of stavudine lipospheres.
Figure 3. Percent drug release from lipospheres prepared with Dynasan 116 (D6X9) and Compritol® 888 ATO (CmpX5) batch.
particle size and percent drug entrapment of lipospheres. As lipid concentration was increased higher drug loading of stavudine could be obtained. Plurol® Oleique CC 497 gave desirable results at lower concentration, while out of three hydrophilic surfactants use of sodium cholate resulted in higher drug entrapment and smaller particle size. The rate of stirring and rate of addition of water phase affected the size distribution of lipospheres.
For lipospheres prepared using trimyristin as lipid matrix and sodium cholate as stabilizer (6:1, w/w), and 5% w/w Dynasan 116/sodium cholate at 6000rpm stirring speed resulted in the production of spherical particles, with good percentage of recovery (90%, w/w) a mean diameter of 25µm and a narrow size distribution was obtained. In the case of lipospheres prepared by using stearic acid,
Tripalmitin, Precirol® ATO 5 resulted in irregular shape, low drug entrapment and sticky end product.
Compritol® 888 ATO lipospheres showed in vitro drug release with initial burst release of around 40% in first hour followed by slow release with around 63% at 8h whereas 77.5% and 66.72%
of drug was released at 24 h from lipospheres prepared with Compritol® 888 ATO and Dynasan 116 respectively (Fig 3). Stearic acid, Dynasan 118 and 114
CONCLUSION
Thus lipospheres of stavudine have been successfully developed. Nature of lipid, concentration of surfactants and rate of stirring speed were the key variables in formulation of lipospheres. The formulation and preparation of stavudine lipospheres were optimized using Table 2: Effect of four independent variables on particle size and drug entrapment of lipospheres.
Dependent variables Lipospheres batches Independent variables
A B C D Particle size (µm) % drug entrapment Dynasan 116
D6X1 1 1 1 1 49±3 25±0.90
D6X2 1 2 2 2 30±5 43±0.59
D6X3 1 3 3 3 35±2 40±0.67
D6X4 2 1 2 3 40±3 45±0.44
D6X5 2 2 3 1 40±3 45±0.44
D6X6 2 3 1 2 50±2 30±0.36
D6X7 3 1 3 2 10±5 25±0.12
D6X8 3 2 1 3 20±6 52±0.35
D6X9 3 3 2 1 30±8 53±0.12
Compritol® ATO 888
CmpX1 1 1 1 1 10±5 30±0.11
CmpX2 1 2 2 2 20±3 25±0.32
CmpX3 1 3 3 3 10±6 32±0.80
CmpX4 2 1 2 3 20±5 37±0.30
CmpX5 2 2 3 1 20±4 40±0.12
CmpX6 2 3 1 2 10±2 26±0.70
CmpX7 3 1 3 2 25±2 31±0.69
CmpX8 3 2 1 3 10±3 33±0.60
CmpX9 3 3 2 1 20±5 25±0.96
a Taguchi orthogonal experimental design.
Type of lipid, drug: lipid molar ratio, and concentration of surfactant has a significant influence (P < 0.05) on the particle size and drug entrapment efficiency. The optimum level of parameters was established as lipids, drug:
lipid molar ratio, Plurol® Oleique CC 497 and sodium cholate concentration, as co-surfactant with regard to optimum particle size and maximum entrapment efficiency.
ACKNOWLEDGEMENTS
The authors are thankful to the Colorcon Asia Pvt. Ltd. and Sasol GmBH, Germany
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